JP2838084B2 - Primers for the detection of HIV-1 - Google Patents

Abstract

The present invention provides improved primers for the polymerase chain reaction (PCR) amplification of a nucleic acid sequence from the gag gene of the human immunodeficiency virus type 1 (HIV-1). The primers and amplification methods of the invention enable the detection of all HIV-1 group M isolates with nearly uniform efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the fields of molecular biology and nucleic acid chemistry. More specifically, the present invention relates to methods and reagents for detecting human immunodeficiency virus type 1 (HIV-1). Thus, the invention applies generally to medicine, in particular to the field of medical diagnostics, and to the field of molecular biology.

[0002]

BACKGROUND OF THE INVENTION Methods for amplifying particular sequences of nucleic acids, particularly the polymerase chain reaction (PCR) invention, allow for the rapid detection of nucleic acids present in a sample, which are hitherto undetectable in small amounts. (US Patent 4,
683,195; 4,683,202; and 4,965,188). The development and application of PCR has been extensively described in the literature.

For example, a wide range of PCR-related topics are described in the PCR Technology-principles and applications fo
r DNA amplification, 1989, (ed. HAErlich) Stockt
onPress, New York, NY; PCR Protocols: A guide to
methods and Applications, 1990 (ed. MAInnis et al.,) Academic Press, San Diego, CA; and PCR Stra
tagies, 1995, (ed. MAInnis et al.) Academic Press,
Discussed in San Diego, CA. Commercial providers, such as Perkin Elmer (Norwalk, CT), market PCR reagents and publish PCR protocols.

The use of PCR and probe hybridization to amplify and detect HIV-1 nucleic acids is described in Kwok, 1992, Ann. Med. 24 : 211-214;
utlee et al., 1991, Mol. Cell. Probes 5 : 241-259. PCR-based HIV-1 detection assays are described, for example, in US Pat. Nos. 5,008,182 and 5,176,775; Kellogg and Kwok, 1990,
PCR Protocols: A Guide to Methods and Application
s, (ed.Innis et al.), Academic Press, SanDiego, CA
: 337-347; Holodniy et al., 1991, J. Inf.Dis. 163 :
802-865; Jockson et al., 1991, AIDS 5 : 1463-1467
And Mulder et al., 1994, J. Clin. Microbiol. 32
(2): described in 292-300.

[0005] Commercially available for amplification and detection of HIV-1
Kits are commercially available from Hoffmann-La Roche (Nutley, NJ).
Have been. Amplicor^TM HIV-1 Test is an HIV-1 pro
An in vitro assay for the detection of viral DNA.
You. AMPLICOR HIV-1 MONITOR ^TM Test is HIV-1
In vitro assay for quantification of RNA. A
Mmplder et al., 1994,
 J. Clin. Microbiol.32 (2): described in 292-300,
Referred to herein as Amplicor HIV-1 primers
Primer pairs SK462 (SEQ ID NO: 5) and SK43
1 (SEQ ID NO: 6) to amplify the HIV-1 nucleic acid.

[0006] HIV-1 shows considerable genomic sequence variability. Phylogenetic analysis of the nucleic acid sequences of the HIV-1 gag and env genes is described in Myers et al., 1993, Human Retrovi.
rusand AIDS 1993, Los Alamos National Laboratory,
Los Alamos, NM (incorporated herein by reference)
It is described in. Within the M group, subtypes AJ have been identified.

[0007] Conventional techniques of molecular biology and nucleic acid chemistry known to those skilled in the art are described in the literature. For example, Sambrook et al., 1989, Molecular Cloning-A Labor
atoryManual, Cold Spring Harbor Laboratory, Cold S
pring Harbor, New York; Oligonucleotide Synthesis
(MJGait, ed., 1984); Nucleic Acid Hybridizatio
n (BDHames and SJ Higgins. eds., 1984); and a series of Methods in Enzymology (Academic Press, Inc.).

[0008] Many human immunodeficiency viruses that cannot or cannot be amplified effectively using the gag gene primers previously described, particularly the Amplicor HIV-1 primers, SK462 (SEQ ID NO: 5) and SK431 (SEQ ID NO: 6). Type 1 (HIV-1) Group M isolates have been identified. These isolates exhibit sequence denaturation not previously found in the region encompassing the primer binding site of the Amplicor HIV-1 primer.

It is an object of the present invention to provide, in addition to all isolates that can be amplified by Amplicor HIV-1 primers, improved primers that allow for efficient amplification from those newly expressed isolates. Is the purpose. In addition, the primers of the present invention allow amplification from all known HIV-1 group M isolates with new constant efficiency. One aspect of the invention relates to polymerases of the region of the gag gene from HIV-1 Group M isolates from subtypes AG with new constant efficiency and without concurrent amplification of non-target sequences. An improved oligonucleotide primer that allows for strand reaction (PCR) amplification.

In particular, the present invention relates to a human immunodeficiency virus type 1 (HIV-1) selected from the group consisting of SKCC1 (SEQ ID NO: 3) and SKCC3 (SEQ ID NO: 4).
Oligonucleotide primers for nucleic acid amplification. Preferably, each of the primers is SK
145 (SEQ ID NO: 1) and SKCC1 (SEQ ID NO: 3)
45 (SEQ ID NO: 1) and a pair of oligonucleotide primers consisting of SKCC3 (SEQ ID NO: 4).

In a further embodiment, the paired primers are oligonucleotide primers SK145
(SEQ ID NO: 1), SKCC1 (SEQ ID NO: 3) and SK1
A set of oligonucleotide primers or oligonucleotide primers SK consisting of 45M2 (SEQ ID NO: 2)
Primer SK145M in a set of oligonucleotide primers consisting of 145 (SEQ ID NO: 1), SKCC3 (SEQ ID NO: 4) and SK145M2 (SEQ ID NO: 2)
2 (SEQ ID NO: 2).

[0012] A further aspect of the present invention comprises performing a PCR using the primers of the present invention.
An improved method for amplifying a region of the gag gene from the V-1 group M subtype. A further aspect of the present invention relates to a kit comprising the amplification primer of the present invention. These kits can include additional reagents, such as a detection probe, or one or more amplification reagents, such as a polymerase, a buffer, and a nucleoside triphosphate.

To aid in understanding the present invention, some terms are defined below. The terms "nucleic acid" and "oligonucleotide" are defined as polydeoxyribonucleotides (2
-Deoxy-D-ribose), polyribonucleotides (including D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base or a modified purine or pyrimidine base. There is no intended distinction in length between the terms "nucleic acid" and "oligonucleotide",
And those terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, the terms are used for double-stranded and single-stranded DN
A, as well as double- and single-stranded RNA.

Oligonucleotides are described in Narang et al., 197
9, Meth. Enzymol. 68 : 90-99 phosphotriester method; Brown et al., 1979, Meth. Enzymol. 68 : 109-151.
Prepared by any suitable method, including cloning and restriction enzyme digestion of the appropriate sequence, and direct chemical synthesis according to the solid support method of U.S. Pat. No. 4,458,066. .
For a review of synthesis methods, see Goodchild, 1990, Bioconjugate Chem.
istry 1 (3): provided at 165-187.

The term "hybridization" refers to the formation of a complex by two single-stranded nucleic acids by complementary base pairing. Hybridization can occur between sufficiently complementary nucleic acid strands or between "substantially complementary" nucleic acid strands that include a minor region of mismatch.
Conditions under which only sufficiently complementary nucleic acid strands will hybridize are referred to as "stringent hybridization conditions" or "sequence-specific hybridization conditions." Stable complexes of substantially complementary sequences can be achieved under low stringency hybridization conditions; the degree of mismatch that can be tolerated can be adjusted by appropriate adjustment of hybridization conditions.

Those of skill in the nucleic acid arts will be able to determine many variables, including oligonucleotide length and base pair concentration, ionic strength, and the incidence of mismatched base pairs, experimentally, according to guidance provided by the art. Can be used to determine the stability of the complex (eg, Sambro
ok, etc., 1989, Molecular Cloning-A Laboratory Manu
al, Cold Spring Harbor Laboratory, Cold Spring Har
bor, New York; and Wetmur, 1991, Critical Reviews
in Biochem. and Mol. Biol. 26 (3/4): 227-259).

The term "primer" refers to a condition under which the synthesis of a primer extension product complementary to a nucleic acid strand is induced, ie, in a suitable buffer, four different nucleoside triphosphates and a polymer. Can act as a starting point for DNA synthesis in the presence of an agent for DNA synthesis (eg, DNA polymerase or reverse transcriptase) and at an appropriate temperature.
An oligonucleotide is either natural or synthetic. The primer is preferably a single-stranded oligodeoxyribonucleotide. The appropriate length of a primer depends on the intended use of the primer, but typically ranges from 15 to 35 nucleotides.

[0018] Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template. The primer has no effect on the exact sequence of the template nucleic acid, but should be sufficiently complementary to hybridize with the template. Primers can include additional features that allow for the detection or immobilization of the primer, but that do not alter the basic properties of the primer that serve as a starting point for DNA synthesis. For example, primers can include additional nucleic acid sequences at the 5 'end that do not hybridize to the target nucleic acid, but facilitate cloning of the amplified product. The region of the primer that is sufficiently complementary to the template to hybridize is referred to herein as a hybridizing region.

As used herein, "upstream" primer refers to a primer whose extension product is a subsequence of the coding strand.
By "downstream" primer is meant a primer whose extension product is a subsequence of the complementary non-coding strand. The terms "target sequence,""targetregion," and "target nucleic acid" refer to a region of a nucleic acid that is to be amplified, detected, or otherwise analyzed.

As used herein, a primer is one in which the number of mismatches between the primer and the target sequence is between the primer and non-target sequences that may be present in the sample. If it is less than the number of mismatches, it is "specific" for the target sequence.

Hybridization conditions can be selected that result in the formation of a stable complex only if the number of mismatches present is not greater than the number of mismatches between the primer and the target sequence. Under such conditions, the target-specific primer can form a stable complex only with the target sequence. Thus, the use of target-specific primers under appropriate stringent amplification conditions allows for specific amplification of the sequence containing the target primer binding site. Similarly, the use of target-specific probes under appropriate stringent hybridization conditions allows for the detection of specific target sequences.

The term "amplification reaction mixture" refers to a solution containing the reagents necessary to carry out the amplification reaction, and typically contains primers, thermostable D
Contains NA polymerase, dNTPs, and divalent metal cations. A reaction mixture is referred to as "complete" if it contains all the reagents necessary to carry out the reaction,
And if it contains only a subset of the necessary reagents,
It is referred to as "incomplete."

For convenience, storage stability, or to allow for application-dependent adjustment of component concentrations, the reaction components are routinely stored as a plurality of separate solutions, each containing a subset of all components. It will be understood by those skilled in the art that storage and mixing of the reaction components prior to the reaction to form a complete reaction mixture. Further, it will be appreciated by those skilled in the art that the reaction components are separately packaged for commercial sale, and that useful commercial kits can include any subset of the reaction components, including the primers of the present invention. .

HIV-1 Amplification Primers The primers of the present invention allow for amplification of nucleic acids from the M subtype of the HIV-1 group. The primers of the present invention include group M, including newly discovered isolates.
From all isolates of subtypes AG belonging to
It shows a significant improvement over previously described primers in allowing amplification of the g gene nucleic acid with constant efficiency from almost any region. The nucleotide sequence of the primer is provided below,
Here, the arrangement is shown from left to right in the 5 'to 3' direction.

[0025]

[Table 1]

[0026] The downstream primer of the present invention can be used with any of the upstream primers disclosed herein. The downstream primer of the present invention can be used preferably with the upstream primer SK145 (SEQ ID NO: 1) and optionally together with the upstream primer SK145M2 (SEQ ID NO: 2). The upstream primer SK145 (SEQ ID NO: 1) was obtained from Kellogg and Kwok, 1990, PCT Protocols:
A Guide to Methods and Applications, (ed. Innis et al.), Academic Press, San Diego, CA: 337-347. The second upstream primer, SK145M2
(SEQ ID NO: 2) hybridizes in the same region as SK145 (SEQ ID NO: 1), but is designed to more closely match the nucleotide sequence of certain HIV-isolates of subtypes A and E. I have. As shown in the examples, the use of both upstream primers can help equalize the efficiency of amplification of particular subtypes.

Amplification Amplification is performed under significantly more stringent conditions that allow amplification of the M subtype of the HIV-1 group, but restore amplification of non-target sequences. Preferred amplification reaction conditions are
Mulder et al., 1994, J. Clin. Microbiol. 32 (2): 292.
-300 and in the product insert of the AMPLICOR HIV-1 MONITOR Test. The exact conditions are not a critical aspect of the present invention. Optimization of amplification conditions is usually
It can be performed based on the guide provided herein.

The primer and the method of the present invention can be used for HIV-
1 can be used to detect either proviral DNA or HIV-1 RNA. Amplification of RNA using reverse transcription / polymerase chain reaction (RT-PCR) is well known in the art and is described in U.S. Patent Nos. 5,322,770 and 5,310,652.
No .; Myers and Gelfand, 1991, Biochemistry 30 (31)
: 7661-7666; and Young et al., 1993, J. Clin. Micro
biol. 31 (4): 882-886. HIV-
1 RT-PCR amplification of RNA is described by Mulder et al., 1994,
J. Clin. Microbiol. 32 (2): 292-300 and Holodniy et al., 1991, J. Inf. Dis. 163 : 802-865.

Suitable sample separation methods for the amplification of HIV-1 DNA and RNA have been described in the literature.
The particular method used is not a critical aspect of the present invention. One skilled in the art can select and optimize an appropriate sample preparation method based on the guides provided herein. A preferred sample preparation method for use in detecting HIV-1 proviral DNA is Casareale
Et al., 1992, PCR Methods and Applications 2 : 149
-153 and Butcher and Spadoro, 1992, Clin.Immunol.Ne
wsletter 12 : 73-76.

A preferred sample preparation kit for the detection of HIV-1 proviral DNA is Amplicor HIV-1 T
Commercially available as part of est. HIV-1 in plasma
A preferred sample preparation method for use in detecting RNA is Mulder et al., 1994, J. Clin. Microbiol. 32 (2)
: 292-300. A preferred sample preparation kit for the detection and / or quantification of HIV-1 RNA is commercially available as part of the AMPLICOR HIV-1 MONITOR Test.

Analysis of Amplified Product The amplification primers and methods of the present invention are suitable for any use with the amplified nucleic acid. For example, cloning and / or sequencing of the HIV-1 sequence is facilitated by use of the primers of the invention. PC
Methods for detecting R-amplified nucleic acids are well known in the art. The method used to analyze the amplified nucleic acid is not a critical aspect of the invention,
And any suitable method can be used. Preferably, amplification of HIV-1 RNA is used as described in the examples to quantify viral load.

Examples of methods for detecting the amplified nucleic acid include analysis of the amplification product by gel electrophoresis and detection by hybridization with a complementary oligonucleotide probe. Suitable assay formats for detecting target-probe hybridization are well known in the art and include dot-blot and reverse dot-blot assay formats.

In the dot-blot format, the amplified target DNA is immobilized on a solid support, for example, a nylon membrane. The membrane-target complex is incubated with the labeled probe under appropriate hybridization conditions, unhybridized probe is removed by washing under appropriate stringent conditions, and the membrane is bound to the bound probe. Is monitored for the presence of Dot-blot detection of PCR amplification products is described, for example, in Saiki et al., 1986, Nature 324 : 163-166 and U.S. Patent No. 5,468,613.

In the reverse dot-blot format, the probe is immobilized on a solid support, eg, a nylon membrane, and the amplified target DNA is labeled. Target DNA
Is typically labeled during amplification by incorporation of labeled primers. One or both primers can be labeled. The membrane-probe complex is incubated with the labeled and amplified target DNA under appropriate hybridization conditions, unhybridized target DNA is removed by washing under appropriate stringent conditions, and then A filter is monitored for the presence of bound target DNA. Reverse dot-blot methods are described, for example, in Saiki et al., 1989, Proc.
atl.Acad.Sci. USA 86 : 6230 and U.S. Pat.
No. 468,613.

Alternatively, the reverse dot-blot assay can be performed using a solid support having multiple probe hybridization sites or wells. For example, microwell plates are particularly useful in large-scale clinical applications of the methods of the present invention. Probes can be immobilized on microwell plates by passive binding or through protein intermediates, such as bovine serum albumin, which attach to the microwell plates (see Tung et al., 1991, Bioconjugate Chem. 2 : 464-465). Thing).

The reverse dot-blot method performed in microwell plates is described in US Pat.
2,829; Loeffelholz et al., 1992, J. Clin. Micr
obiol. 30 (11): 2847-2851; Jackson et al., 1991, A
IDS 5 : 1463-1467; Mulder et al., 1994, J. Clin. Mi
crobiol. 32 (2): 292-300; the Amplicor HIV-1Tes
t Product insert; and AMPLICOR HIV-1 MONITOR Test product insert.

Preferably, the detection and / or quantification of the amplified product is performed by Amplicor HIV-1 Test or AMPLICOR H
Using the reagents and protocol of IV-1 MONITOR Test,
This is performed by hybridization with an oligonucleotide probe immobilized on a microwell plate. The use of the method of the invention to quantify HIV-1 RNA is also described in the examples. Alternatively, BSA
The bonded probe is bound to the magnetic particles; The bound probe is hybridized in solution to the labeled amplification product, and the resulting is magnetically removed from solution. The magnetically immobilized hybridization complex is detected as in the method above.

Another suitable assay, referred to as the 5'-nuclease assay, is described in US Pat. Nos. 5,210,015 and 5,487,972, and Holland et al., 1988, Proc. Natl. Acad.Sci. USA 88
: 7276-7280. In a 5'-nuclease assay, a modified, labeled detection probe is added to the reaction mixture during amplification to prevent the probe from acting as a primer for DNA synthesis.

During each synthesis step, ie, during primer extension, any probe that hybridizes to the target DNA will have a DNA polymerase, such as Taq D
It is degraded by the 5 'to 3' exonuclease activity of NA polymerase. Next, degradation products from the probe are detected. Thus, the presence of the probe degradation product indicates that hybridization between the probe and the target DNA has occurred and that an amplification reaction has occurred. U.S. Pat. Nos. 5,491,063 and 5,571,6
No. 73 describes an improved method for detecting probe degradation that occurs simultaneously with amplification.

The above assay formats typically use labeled oligonucleotides to facilitate detection of the hybrid complex. Oligonucleotides, spectroscopy,
It can be labeled by incorporating a label that can be detected by photochemical, biochemical, immunochemical or chemical means. Useful labels include ³² P, fluorescent dyes, electron crowding reagents, enzymes (E
(As commonly used in LISAS), biotin, or haptens and proteins for which antisera or monoclonal antibodies are available. The labeled oligonucleotides of the present invention can be synthesized and labeled using the techniques described above for synthesizing oligonucleotides.

Another method for detecting amplification of HIV-1 nucleic acid by monitoring the increase in the total amount of double-stranded DNA in the reaction mixture is described in Higuchi et al., 1
992, Bio / Technology 10 : 413-417: Higuchi et al.,
1993, Bio / Technology 11 : 1026-1030; and EP-A-512,334. Detection of double-stranded target DNA relies on the enhanced fluorescence exhibited when ethidium bromide (EtBr) and other DNA-binding labels are bound to double-stranded DNA. A DNA binding label is added to the amplification reaction mixture. Amplification of the target sequence results in an increase in double-stranded DNA, which results in a detectable increase in fluorescence.

The present invention also relates to kits, ie, multi-container units comprising useful components for practicing the present invention. Useful kits include primers for PCR amplification of HIV-1 nucleic acids. The kit can also include a means for detecting the amplified HIV-1 nucleic acid, eg, an oligonucleotide probe. In many cases, the probe is immobilized on a suitable support membrane. Other optional components for the kit include, for example, reagents for catalyzing the synthesis of primer extension products, substrate nucleoside triphosphates, means used to label (eg,
Avidin-enzyme conjugates and enzyme substrates and, if the label is biotin, a chromogen), suitable buffers for PCR or hybridization reactions, and instructions for practicing the present invention.

[0043]

The following examples of the present invention are illustrative and not limiting. Example 1 . Generation of Quantitative Standards Quantification of viral load, such as that performed using the AMPLICOR HIV-1 MONITOR Test, is performed using a quantification standard (QS) that is amplified using the same primer pair but detected using another probe. ) Is used. QS is added to the test sample at a known concentration to provide a known control signal. For a wide range of target concentrations, the signal generated from the amplified target or amplified QS is proportional to the amount present. Target copy number is known QS
Calculated from the comparison of the signal generated from the amplification of the unknown target to the signal generated from.

The primer of the present invention is AMPLICOR HIV-1 M
Amplify a region that is not sufficiently included in the QS included in the ONITOR Test (the Amplicor QS). Therefore, the new QS
Was made for use with the primers of the present invention. The new QS comprises SKCC1 (SEQ ID NO: 3) and S
Ampl to include the binding site for KCC3 (SEQ ID NO: 4)
It was constructed from Amplicor QS by extending the icor QS sequence. The resulting QS is AMPLICOR HIV-1 M
It can be detected using the QS-specific probe used in the ONITOR Test. Creation of a plasmid containing a novel QS sequence from which QS RNA is transcribed can be performed using the following standard techniques.

Amplicor QS obtained from the AMPLICOR HIV-1 MONITOR Test was obtained under the conditions substantially as described in Example 2 below, under the conditions of SK145 (SEQ ID NO: 1) and SK151.
(SEQ ID NO: 7). The amplification is SK
A DNA product is generated that contains the primer binding sites for 145 (SEQ ID NO: 1) and SK151 (SEQ ID NO: 7) and retains the internal sequence that contains the binding site for the QS-specific probe.

Next, the obtained amplified product was subjected to SK
It is extended to include the primer binding site of CC1 (SEQ ID NO: 3) and a linker is added to allow cloning of the product. This is achieved by reamplifying the product with SK145 (SEQ ID NO: 1) extended at the 5 'end to include a HindIII linker and SKCC1 (SEQ ID NO: 3). Suitable amplification conditions are described in Kellogg and Kwok, 1990, PCR Protocols: A Gu
id to Methods and Applications, (ed.Innis et al.,),
Academic Press, San Diego, CA: 337-347, except that a lower annealing / extension temperature (eg, 42 ° C.) is used to allow hybridization of SKCC1 (SEQ ID NO: 3). .

Next, the obtained amplified product was subjected to SK
It is further extended to include the primer binding site of CC3 (SEQ ID NO: 4) and a second linker is added at the other end to allow cloning of the product. This was extended at the 5 'end to include the HindIII linker as described above, with SKCC3 (SEQ ID NO: 4) extended at the 5' end to include the XBaI linker, using the same amplification conditions described above. SK145 (SEQ ID NO: 1).

Next, the amplified product is inserted into a plasmid. The amplified DNA and plasmid pS
P64 (Pramega, Madison, WI) is digested separately with HindIII and XBaI and then ligated using standard methods. Competent cells are transformed with the recombinant plasmid and clones containing the correct insert are obtained. The cloned insert in the resulting recombinant plasmid should be sequenced to determine that no mutation has been introduced in the primer or probe binding site. QS RNA, MEGAscript ^TM S
QS using P6 kit (Ambion, Inc., Austin, TX)
Transcribe from a recombinant plasmid containing the sequence.

Example 2 Quantification of HIV-1 RNA This example describes the evaluation of the relative efficiency of amplification from various HIV-1 isolates. For comparison, the amplification was also AM
The test was performed using the PLICOR HIV-1 MONITOR Test Kit. H
IV-1 isolates were obtained from HIV-1 positive clinical samples. The region of the gag gene was cloned and sequenced using standard techniques and the cloned HI
The subtype of V-1 was determined based on its sequence.
Many of these HIV-1 isolates have been discovered to be novel. For this example, based on the nucleotide sequence, specific isolates were selected that were predicted to be problematic for the AMPLICOR HIV-1 MONITOR Test.
In addition, isolates showing sequence variation present in the group M subtype were used.

Plasmids containing the region of the HIV-1 gag gene from individual isolates of the target nucleic acid were made, and the HIV-1 RNA template was cloned from Holodniy et al., 1991, J. Inf. Dis. 163 : 802-. 865
Were transferred substantially as described in Stock solutions of individual templates were prepared and template concentrations were assayed. Stock solutions were diluted based on the relative concentrations evaluated so that the concentration of template added to each reaction was the same. The absolute number of copies of the template added to the reaction was about 4000-8000.

Quantification QS as described in Standard Example 1 was introduced into individual reaction mixtures at a known concentration, typically 100 copies / reaction. Primer reactions AF were performed using the following primer combinations:

[0052]

[Table 2]

All primers were biotinylated at the 5 'end to allow detection in a reverse dot-blot microwell plate format. The sequences of additional primers not described above are provided below, which are 5 '
3 'from the side.

[0054]

[Table 3]

[0055] The amplification amplification reaction A, was performed using AMPLICOR HIV-1 MONITOR Test reagents and conditions. Amplification reaction B was performed in a 100 μl volume containing the following reagents:

[0056]

[Table 4]

Amplification reactions C and D were performed under conditions substantially as used in reaction B, but with the following changes:

[0058]

[Table 5]

Amplification reactions E and F were performed under conditions substantially as used in reactions C and D, except that glycerol was used at a concentration of 10%. Slight differences in the reaction mixtures were due to prior optimization of amplification conditions for individual primer pairs. Amplification reactions BF were performed on a TC9600 DNA thermal cycler (Perkin Elmer, Norwalk, Conn.) Using the following temperature profile:

[0060]

[Table 6]

Increase by probe hybridization
Detection of Broadened Products The amplified products were detected using the reagents and protocol of the AMPLICOR HIV-1 MONITOR Test. Estimated initial target concentrations were calculated as described herein.

Results In the AMPLICOR HIV-1 MONITOR Test quantification method, the initial target concentration was estimated from a comparison of the signal generated after amplification of the target to the signal generated after amplification of a known concentration of QS. It is. Since the known concentration of QS is the preamplification value, but the signal being compared is the post-amplification signal, changes in the relative efficiency of amplification will affect the estimation of the initial concentration of the unknown target. Would.
AMPLICORHIV-1 MONITOR Test Quantification is calibrated based on HIV-1 subtype B amplification efficiency. Other HIV
The -1 subtype can be amplified with low efficiency and consequently the estimated target concentration is known to be an underestimate of the true concentration.

In the experiment of the present invention, the target RNA was
Known concentrations were added to each reaction. Thus, the relative amplification efficiencies for individual isolates can be determined by comparing the assessed target concentrations. AMPLICOR
HIV-1 MONITOR Test Quantification is HIV-1 subtype B
The subtype B is calibrated based on the amplification efficiency of
The estimated target concentration of (clone 105-1) was used as a control. For individual isolates, the estimated target concentration for subtype B (clone 105-1) was estimated for the isolate to provide a measure of relative amplification efficiency. Divided by the target concentration.
Their relative amplification efficiencies are reported in the table below.
The entry "---" indicates that no reaction was performed.

[0064]

[Table 7]

The above results indicate that AMPLICOR HIV-1 MONITOR Tes
t (Reaction A) shows that different HIV-1 isolates were amplified with significant variation in efficiency. Some isolates, such as subtype E isolate, clone 1
10-5 was amplified with an efficiency at least about 500-fold lower than that of subtype B, clone 105-1.

Although not all isolates were tested, primer pairs SK145 (SEQ ID NO: 1) and SK1
The use of 51 (SEQ ID NO: 7) (Reaction B) appeared to significantly improve the amplification efficiency uniformity. However, the subtype E isolate, clone 110-5, was still amplified, with an efficiency approximately 500 times lower than that of subtype B, clone 105-1.

SK145 (SEQ ID NO: 1) and SKCC1
The use of (SEQ ID NO: 3) (reaction C) is within about three times the efficiency of amplification of subtype B, clone 105-1
The amplification of all isolates, including the subtype E isolate, clone 110-5, was enabled. Addition of SK145M2 (SEQ ID NO: 2) (Reaction D) further improved the efficiency of amplification of isolate 110-5 to be substantially equivalent to that of the subtype B control strain.

Similarly, SK145 (SEQ ID NO: 1) and S
The use of KCC3 (SEQ ID NO: 4) (Reaction E) was subtype E isolate, clone 110-, with an efficiency within about 5 times that of the amplification of subtype B, clone 105-1.
5 allowed the amplification of all isolates. SK
Addition of 145M2 (SEQ ID NO: 2) (Reaction F) further improved the efficiency of amplification of isolate 110-5 to be substantially equivalent to that of the subtype B control strain.

Example 3 HIV-1 in clinical samples
Quantification In this example, 30 clinical samples from seropositive patients from Senegal were assayed for the presence of HIV-1 RNA. No subtype present in the clinical sample was determined. However, because subtypes A and D are common in this region of Africa, some of the clinical samples are not amplified or
It was predicted that amplification would not be effective using the AMPLICOR HIV-1 MONITOR Test.

A sample was prepared as follows. Plasma samples (80-250 μl) were placed in a 1.5 ml conical centrifuge tube at 0.25% (w / v) red Estapo.
r polystyrene microspheres (Bangs Laboratories, Inc.,
Carmel, IN) combined with 20 μl and 25,3
Centrifuged at 00 × g for 1 hour at 4 ° C. Aspirate the supernatant and pellet the pellet with 250 μl lysis buffer (50 μl lysis buffer is 6.7 μl 300 / μl
RNasin (Promega, Madison, WI); 0.67μ
1 of 100 mM DTT; 2 μl of 10% NP40 (Pi
erce, Rockford, IL); 0.25 μl of 4 mg / ml poly-
rA RNA; and 40.4 μl of Depc-treated water). The pellet was incubated at room temperature for at least 15 minutes and spun to ensure mixing.

Amplification was performed in a 100 μl reaction containing 50 μl of a 2 × mixture of amplification reagents and 50 μl of virus lysate solution formulated to give a final reagent concentration as described above. Approximately 100 copies of the appropriate QS were added to each reaction as part of the reagent mixture. Detection of the amplified product was performed as described above for AMPLICOR HIV-1.
The test was performed using the reagents and protocol of MONITOR Test. Amplification of individual samples was performed using the following primer combinations.

[0072]

[Table 8] The results, expressed in copies of HIV-1 template / ml plasma, are summarized below.

[0073]

[Table 9]

The above results show that the primers of the invention, combinations B and C, enabled amplification from many clinical samples. Primer combinations B and C allowed amplification from all but one of the 30 samples. In contrast, AMPLICOR HIV-1 MONIT
The OR Test failed to amplify 6 of the 29 samples. Amplification of sample MIH053 using the AMPLICOR HIV-1 MONITOR Test resulted in a strong target signal, but failed to generate a QS signal. Therefore, the sample could not be quantified and is indicated as "+" in the table above.

Further, for a significant number of samples,
The copy number estimated using either primer combination B or C was significantly higher than the copy number estimated using the AMPLICOR HIV-1 MONITOR Test. Based on the amplification efficiency variability shown in Example 2 above, AMPLICOR HIV
-1 Low estimate of template concentration obtained using MONITOR
Probably resulted in significantly lower amplification efficiency. Virus R
NA was not detected in sample MIN067.
However, it is not known whether this is due to apparent variability in the target sequence, which is hindered by either primer or probe hybridization, or by some other cause.

Sequence Listing (2) Information on SEQ ID NO: 1 (i) Sequence characteristics: (A) Length: 30 base pairs (B) Type: nucleic acid (C) Number of chains: single strand ( D) Topology: linear (ii) Sequence type: DNA (genome) (xi) Sequence: SEQ ID NO: 1: AGTGGGGGGA CATCAAGCAG CCATGCAAAT 30

(2) Information on SEQ ID NO: 2 (i) Sequence characteristics: (A) Length: 30 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: linear (ii) Sequence type: DNA (genome) (xi) Sequence: SEQ ID NO: 2: AGTGGGGGGA CACCAGGCAG CAATGCAAAT 30

(2) Information on SEQ ID NO: 3 (i) Sequence characteristics: (A) Length: 28 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: linear (ii) Sequence type: DNA (genome) (xi) Sequence: SEQ ID NO: 3: TACTAGTAGT TCCTGCTATG TCACTTCC 28

(2) Information on SEQ ID NO: 4 (i) Sequence characteristics: (A) Length: 26 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: linear (ii) Sequence type: DNA (genome) (xi) Sequence: SEQ ID NO: 4: TGAAGGGTAC TAGTAGTTCC TGCTAT 26

(2) Information on SEQ ID NO: 5 (i) Sequence characteristics: (A) Length: 30 base pairs (B) Type: nucleic acid (C) Number of chains: single strand (D) Topology: linear (ii) Sequence type: DNA (genome) (xi) Sequence: SEQ ID NO: 5: AGTTGGAGGA CATCAAGCAG CCATGCAAAT 30

(2) Information on SEQ ID NO: 6 (i) Sequence characteristics: (A) Length: 27 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: linear (ii) Sequence type: DNA (genome) (xi) Sequence: SEQ ID NO: 6: TGCTATGTCA GTTCCCCTTG GTTCTCT 27

(2) Information on SEQ ID NO: 7 (i) Sequence characteristics: (A) Length: 27 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: linear (ii) Sequence type: DNA (genome) (xi) Sequence: SEQ ID NO: 7: TGCTATGTCA CTTCCCCTTG GTTCTCT 27

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Shirley E. Quok United States of America, California 94583, San Ramon, Lomond Circle 611 (72) Inventor Seeda Yul United States of America, California 95104, Cappartino, Lindenbrook Lane 19875 (56) References JP-A-4-152899 (JP, A) JP-A-6-225760 (JP, A) JP-A-8-242898 (JP, A) JP-A-7-502654 (JP, A) JP-A-4 -502251 (JP, A) US Patent 5,665,545 (US, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/00 C12Q 1/70 CA (STN)

Claims (15)

(57) [Claims] 1. SKCC1 (SEQ ID NO: 3) and SKCC
Oligonucleotide primers for amplification of human immunodeficiency virus type 1 (HIV-1) nucleic acid selected from the group consisting of SEQ ID NO: 3 (SEQ ID NO: 4) 2. SK145 (SEQ ID NO: 1) and SKCC
1 (SEQ ID NO: 3). 3. A pair of oligonucleotide primers comprising the pair of oligonucleotide primers according to claim 2 and SK145M2 (SEQ ID NO: 2). 4. SK145 (SEQ ID NO: 1) and SKCC
3 (SEQ ID NO: 4). 5. A pair of oligonucleotide primers comprising the pair of oligonucleotide primers according to claim 4 and SK145M2 (SEQ ID NO: 2). 6. A human immunodeficiency virus type 1 comprising the oligonucleotide primer of claim 1.
(HIV-1) A kit for detecting a nucleic acid. 7. A kit for detecting human immunodeficiency virus type 1 (HIV-1) nucleic acid, comprising the pair of oligonucleotide primers according to claim 2. 8. A kit for detecting a human immunodeficiency virus type 1 (HIV-1) nucleic acid, comprising a set of oligonucleotide primers according to claim 3. 9. A kit for detecting a human immunodeficiency virus type 1 (HIV-1) nucleic acid, comprising the pair of oligonucleotide primers according to claim 4. 10. A kit for detecting a human immunodeficiency virus type 1 (HIV-1) nucleic acid, comprising a set of oligonucleotide primers according to claim 5. 11. SKCC1 (SEQ ID NO: 3) or SKC
A method for amplifying a human immunodeficiency virus type 1 (HIV-1) nucleic acid, comprising performing a polymerase chain reaction with C3 (SEQ ID NO: 4). 12. The method according to claim 12, wherein the polymerase chain reaction is SK14.
The method according to claim 11, which is carried out using SKCC1 (SEQ ID NO: 3) and SKCC1 (SEQ ID NO: 3). 13. The method according to claim 12, wherein the polymerase chain reaction
13. The method according to claim 12, which is performed using K145M2 (SEQ ID NO: 2). 14. The method according to claim 14, wherein the polymerase chain reaction comprises
K145 (SEQ ID NO: 1) and SKCC3 (SEQ ID NO: 4)
The method of claim 11, wherein the method is performed using 15. The method according to claim 15, wherein the polymerase chain reaction comprises
The method according to claim 11, which is carried out using K145M2 (SEQ ID NO: 2).